Ld Phase Research Articles

Crystallization around solid-like nanosized docks can explain the specificity, diversity, and stability of membrane microdomains

To date, it is widely accepted that microdomains do form in the biological membranes of all eukaryotic cells, and quite possibly also in prokaryotes. Those sub-micrometric domains play crucial roles in signaling, in intracellular transport, and even in inter-cellular communications. Despite their ubiquitous distribution, and the broad and lasting interest invested in those microdomains, their actual nature and composition, and even the physical rules that regiment their assembly still remain elusive and hotly debated. One of the most often considered models is the raft hypothesis, i.e., the partition of lipids between liquid disordered and ordered phases (Ld and Lo, respectively), the latter being enriched in sphingolipids and cholesterol. Although it is experimentally possible to obtain the formation of microdomains in synthetic membranes through Ld/Lo phase separation, there is an ever increasing amount of evidence, obtained with a wide array of experimental approaches, that a partition between domains in Ld and Lo phases cannot account for many of the observations collected in real cells. In particular, it is now commonly perceived that the plasma membrane of cells is mostly in Lo phase and recent data support the existence of gel or solid ordered domains in a whole variety of live cells under physiological conditions. Here, we present a model whereby seeds comprised of oligomerised proteins and/or lipids would serve as crystal nucleation centers for the formation of diverse gel/crystalline nanodomains. This could confer the selectivity necessary for the formation of multiple types of membrane domains, as well as the stability required to match the time frames of cellular events, such as intra- or inter-cellular transport or assembly of signaling platforms. Testing of this model will, however, require the development of new methods allowing the clear-cut discrimination between Lo and solid nanoscopic phases in live cells.

Changes in Membrane Organization upon Spontaneous Insertion of 2-Hydroxylated Unsaturated Fatty Acids in the Lipid Bilayer

Recent research regarding 2-hydroxylated fatty acids (2OHFAs) showed clear evidence of their benefits in the treatment of cancer, inflammation, and neurodegenerative disorders such as Alzheimer's disease. Monolayer compressibility isotherms and isothermal titration calorimetry of 2OHFA (C18-C22) in phosphatidylcholine/phosphatidylethanolamine/sphingomyelin/cholesterol (1:1:1:1 mole ratio), a mixture that mimics the composition of mammalian plasma membrane, were performed to assess the membrane binding capacity of 2OHFAs and their natural, nonhydroxylated counterparts. The results show that 2OHFAs are surface-active substances that bind membranes through exothermic, spontaneous processes. The main effects of 2OHFAs are a decrease in lipid order, with a looser packing of the acyl chains, and a decreased dipole potential, regardless of the 2OHFAs' relative affinity for the lipid bilayer. The strongest effects are usually observed for 2-hydroxyarachidonic (C20:4) acid, and the weakest one, for 2-hydroxydocosahexaenoic acid (C22:6). In addition, 2OHFAs cause increased hydration, except in gel-phase membranes, which can be explained by the 2OHFA preference for membrane defects. Concerning the membrane dipole potential, the magnitude of the reduction induced by 2OHFAs was particularly marked in the liquid-ordered (lo) phase (cholesterol/sphingomyelin-rich) membranes, those where order reduction was the smallest, suggesting a disruption of cholesterol-sphingolipid interactions that are responsible for the large dipole potential in those membranes. Moreover, 2OHFA effects were larger than for both lo and ld phases separately in model membranes with liquid disordered (ld)/lo coexistence when both phases were present in significant amounts, possibly because of the facilitating effect of ld/lo domain interfaces. The specific and marked changes induced by 2OHFAs in several membrane properties suggest that the initial interaction with the membrane and subsequent reorganization might constitute an important step in their mechanisms of action.

The effect of hydroxylated fatty acid-containing phospholipids in the remodeling of lipid membranes

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) is an antitumor drug that regulates membrane lipid composition and structure. An important effect of this drug is the restoration of sphingomyelin (SM) levels in cancer cell membranes, where the SM concentration is lower than in non-tumor cells. It is well known that free fatty acid concentration in cell membranes is lower than 5%, and that fatty acid excess is rapidly incorporated into phospholipids. In a recent work, we have considered the effect of free 2OHOA in model membranes in liquid ordered (Lo) and liquid disordered (Ld) phases, by using all-atom molecular dynamics. This study concerns membranes that are modified upon incorporation of 2OHOA into different phospholipids. 2OHOA-containing phospholipids have a permanent effect on lipid membranes, making a Ld membrane surface more compact and less hydrated, whereas the opposite effect is observed in Lo domains. Moreover, the hydroxyl group of fatty acid chains increases the propensity of Ld model membranes to form hexagonal or other non-lamellar structures. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Temperature Dependence of Lo/Ld Domain Thickness and Elasticity by Global Saxs Data Analysis

The spatial organization of lipids in biological membranes plays an important role in diverse cellular processes. Of particular interest are membrane rafts which are considered to enable cellular signaling and transport. Such membrane rafts are currently mimicked by liquid ordered (Lo) domains, observed in several lipid-only mixtures. The physical properties of such Lo domains and the coexisting liquid disordered (Ld) phase are presently not well known. We developed a global small-angle x-ray scattering data analysis for multilamellar vesicles that allows to determine membrane structural parameters and bending fluctuations of coexisting lipid domains. Different scattering length density profiles were used for modeling the lipid bilayers with increasing details and complexity. The technique was applied to mixtures of DOPC/DPPC/Chol and DOPC/DSPC/Chol. We determined membrane thickness as well as bending elasticity for Lo and Ld phases as a function of lipid composition and temperature. In particular, we discuss changes in these parameters upon the transition to a homogeneous phase.

Membranes with Thick, Liquid-Disordered and Thin, Liquid-Ordered Phases are Rare

Lipid membranes containing ternary mixtures of a lipid with a high melting temperature (Tm), a lipid with a low-Tm, and a sterol can phase separate into two coexisting liquid phases over a range of temperatures and compositions (Veatch 2003 Biophys. J.). The two phases are termed liquid-ordered (Lo) and liquid-disordered (Ld). It is generally assumed that the Lo phase is thicker than the Ld phase. We hypothesize that increasing the chain length of the low-Tm lipid and/or decreasing the chain length of the high-Tm lipid yields systems with thicker Ld phases. We measured phase heights of membranes containing high-Tm lipids with saturated tails of 12 to18 carbons, and low-Tm lipids with unsaturated tails of 20 to 24 carbons or phytanoyl chains. Of all these combinations, only one ternary system produced data consistent with thicker Ld than Lo phases: DPPC/Di(22:1)PC/cholesterol. To identify which phase was thicker in each membrane, we first ruptured giant phase-separated vesicles labeled with a fluorescent probe on mica to form supported lipid bilayers. This allowed us to directly compare fluorescence images (to identify Lo and Ld phases) to AFM images (to reports heights) of the same vesicle. We expanded our study to include bilayers with compositions from a recent study from Lin and London (2013 J. Biol. Chem.). These compositions are believed phase separate into thicker Ld nanodomains. To our knowledge, we present the first direct observations of systems in which the thicker liquid phase is not the more ordered phase, showing that height and order need not be correlated.

Area Per Lipid of Membranes from Natural Abundance Solid-State 13C NMR Spectroscopy

The properties of liquid-crystalline membranes vary according to the molecular composition of the lipid bilayer [1]. Structural investigations of lipid membranes using NMR spectroscopy generally require isotopic labeling of the lipids, thereby precluding investigations of complex lipid systems. Combining natural-abundance solid-state 13C magic-angle-spinning (MAS) NMR with a statistical mean torque (MT) model [2] enabled us to obtain the average area per lipid and volumetric bilayer thickness DB for lipid bilayers. The 2D separated local field (SLF) NMR experiment DROSS gives isotropic chemical shifts in the F2 frequency dimension and site-specific dipolar Pake doublets in the F1 dimension [1]. By analyzing the Pake patterns we extracted 13C-1H residual dipolar couplings (RDCs) and calculated segmental |SCH| order parameters. The |SCH| order parameters carry the structural information that aid calculation of and DB using the MT model. Experiments were conducted with DMPC, POPC, and EYSM lipids as single component (liquid-disordered, ld) lipid bilayers as well as with cholesterol. EYSM is more ordered in the ld phase and hence experiences less structural perturbation upon adding cholesterol to form the liquid-ordered (lo) phase relative to POPC. We infer that the cholesterol stiffening effect on lipid bilayers is limited by the maximum volumetric hydrocarbon thickness [2]. Knowledge of structural parameters like and DB is important for molecular dynamics (MD) simulations and provides information about the balance of forces in membrane lipid bilayers [3]. Our studies demonstrate the applicability of solid-state 13C NMR spectroscopy to site-specific structural investigations of complex lipids, where isotope labeling may be prohibitive, allowing structural studies of biologically relevant membrane systems. [1] A. Leftin et al.(2013) JMB425 2973-2987. [2] H. I. Petrache et al.(2000) BJ.79 3172-3192. [3] A. Leftin et al.(2011) BBA1808 818-839.

Protein Partitioning in Liquid-Ordered (LO) / Liquid-Disordered (LD) Domains Depends on Lipid Composition and Protein Shape

The lack of transmembrane proteins partitioned in the current lipid-only models for membrane rafts, i.e. Lo phases, calls for close scrutiny of raft mimetics. Using small angle X-ray scattering (SAXS) and molecular dynamic simulations (MD), we determined structural and elastic parameters (spontaneous curvature, bending rigidity, Gaussian curvature modulus) for coexisting Lo/Ld domains in ternary mixtures of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine/cholesterol (DOPC/DPPC/Chol) and dioleoylphosphatidylcholine/distearoylphosphatidylcholine/cholesterol (DOPC/DSPC/Chol) [1,2]. Substituting these values into theoretical calculations yields the energy penalty upon insertion of transmembrane proteins into Lo and Ld phases, and consequently the preferred partitioning in one of these domains. We discuss our findings for different geometric protein shapes.View Large Image | View Hi-Res Image | Download PowerPoint SlideReferences[1] B. Kollmitzer, P. Heftberger, M. Rappolt, and G. Pabst. Soft Matter (submitted). Preprint: arXiv:1307.0367 [cond-mat.soft].[2] G. Khelashvili, B. Kollmitzer, P. Heftberger, G. Pabst, and D. Harries. J. Chem. Theory Comput. 9, 3866 (2013).This work is supported by the Austrian Science Funds FWF, Project No. P24459.

Cholesterol Binding Drives Partitioning of the Amyloid Precursor C99 Protein Into Liquid Ordered Membrane Domains

The association of the 99 residue transmembrane C-terminal domain (C99) of the amyloid precursor protein with cholesterol-rich membrane domains (“lipid rafts”) is thought to promote cleavage of C99 to release the Alzheimer's disease-promoting amyloid-β (Aβ) polypeptide. Here, we test the hypothesis that C99 has an intrinsic affinity for raft-like liquid ordered (Lo) domains and that its domain preference is regulated by cholesterol binding. Fluorescence microscopy shows that C99 undergoes efficient partitioning into Lo domains relative to the co-existing liquid disordered (Ld) phase in giant unilamellar vesicles, establishing C99 as the transmembrane protein with the highest affinity for Lo domains known to date. Lo partitioning is prevented upon mutating residues that are critical for cholesterol binding to C99, indicating that binding of this sterol is a critical determinant of the phase preference of the protein. Addition of coprostanol, a raft-breaking sterol that can compete with cholesterol for binding to C99, shifts partitioning away from the Lo phase. These findings identify cholesterol binding as a novel mechanism that controls the raft targeting of C99 and suggest that pharmacological inhibition of this process could potentially be exploited as a therapeutic approach to avoid or treat Alzheimer's disease.

Solubilization of Giant Vesicles Composed of Erythrocyte Lipid Extracts and of Ternary Lipid Mixtures by Triton X-100

Detergents are widely used as solubilizing agents of biological membranes. Detergent resistant membranes, called DRMs, are frequently obtained and share similarities in composition and properties with the so-called lipid rafts and with the liquid-ordered phase (Lo) of model membranes. Here we use optical and fluorescence microscopy to study the solubilization process of giant unilamellar vesicles (GUVs) made of erythrocyte lipid extracts (erythro-GUVs) by the commonly used detergent Triton X-100. For comparison, ternary lipid mixtures of POPC, sphingomyelin (SM) and cholesterol are also investigated. Erythro-GUVs do not exhibit macroscopic phase separation, as seen by the homogenous distribution of a fluorescence probe that preferentially partition into the fluid (Ld) phase. However, contact with Triton X-100 immediately causes formation of micrometer-sized bright domains, typical of Lo/Ld phase separation. Then, at Triton X-100 concentrations above its cmc, the fluid part is first detached from the original vesicle and then completely solubilized by Triton X-100. The insoluble vesicle left is only faintly fluorescent and appears to be in the Lo phase. Typically, one third of the erythro-GUV surface area is solubilized. Solubilization of POPC:SM:chol GUVs in a certain composition range follow the same sequence of events. Our results show that Triton X-100 promotes formation of macroscopic domains in both biological and biomimetic membranes, and that the partial solubilization occurs after phase separation, suggesting that the composition of the insoluble membrane fraction is modulated by the choice of the detergent used. Financial support: CNPq, FAPESP and INCT-FCx.

Regulation of Rac translocation and activation by membrane domains and their boundaries

The activation of Rac1 and related Rho GTPases involves dissociation from Rho GDP-dissociation inhibitor proteins and translocation to membranes, where they bind effectors. Previous studies have suggested that the binding of Rac1 to membranes requires, and colocalizes with, cholesterol-rich liquid-ordered (lo) membrane domains (lipid rafts). Here, we have developed a fluorescence resonance energy transfer (FRET) assay that robustly detects Rac1 membrane targeting in living cells. Surprisingly, FRET with acceptor constructs that were targeted to either raft or non-raft areas indicated that Rac1 was present in both regions. Functional studies showed that Rac1 localization to non-raft regions decreased GTP loading as a result of inactivation by GTPase-activating proteins. In vitro, Rac1 translocation to supported lipid bilayers also required lo domains, yet Rac1 was concentrated in the liquid-disordered (ld) phase. Single-molecule analysis demonstrated that translocation occurred preferentially at lo-ld boundaries. These results, therefore, suggest that Rac1 translocates to the membrane at domain boundaries, then diffuses into raft and non-raft domains, which controls interactions. These findings resolve discrepancies in our understanding of Rac biology and identify novel mechanisms by which lipid rafts modulate Rho GTPase signaling.

The Unique Roles of Hybrid Lipids in Lipid Membrane Domain Size and Order

Hybrid lipids (HL) are phospholipids with one saturated and one unsaturated chains. Although it is believed that hybrid lipids can act as linactants (i.e., 2D surfactants) in cell membranes to reduce line tension of lipid domains, as a group, their fundamental roles in membrane heterogeneity have not yet been systematically investigated. In this study, three hybrid lipids of different degrees of chain unsaturation (i.e., 16:0-18:1PC (POPC), 16:0-18:2PC (PLPC), and 16:0-20:4PC (PAPC)) are compared in their abilities to alter the composition, order and compactness of lipid domains. The liquid-ordered and liquid-disordered (Lo+Ld) phase boundaries of HL/di18:0PC(DSPC)/cholesterol systems were determined from giant unilamellar vesicles (GUV). We found that the Lo and Ld lipid domains in PAPC/DSPC/CHOL and PLPC/DSPC/CHOL mixtures are micron-sized, and only POPC/DSPC/CHOL system has nanoscopic domains. The results show that some poly-unsaturated HLs essentially behave like the double-unsaturated di18:1PC (DOPC), and the mono-unsaturated POPC clearly displays both properties of unsaturated lipid and linactant. The obtained phase boundaries indicate that both POPC and PLPC have significant partition in the Lo phases. Our MD simulations reveal that these hybrid lipids decrease the order and compactness of Lo domains. Thus, hybrid lipids distinguish themselves from other lipids in this combined “partitioning and loosening” ability in Lo domains, and this ability could facilitate membrane proteins to partition into lipid-raft-like domains in biomembranes. Our line tension measurement and Monte Carlo simulation both show that even in the best cases, HLs are weak linactants with only modest ability in reducing line tension. Furthermore, the reduction is through two separated mechanisms: one through reducing the compositional differences of Lo and Ld domains, and the other through weak linactant action.

A Systematic Study of Phase Changes Induced by Trans-Membrane Peptide Gramicidin-A in Multi-Component Lipid Membranes

What are the effects of proteins on lipid membrane domains? In order to answer this question, we systematically investigated the phase changes induced by trans-membrane peptide gramicidin-A in 16:0-18:2PC(PLPC)/di18:0PC(DSPC)/cholesterol and 16:0-18:2PC/di16:0PC(DPPC)/cholesterol lipid bilayers. Quaternary giant unilamellar vesicles (GUV) were prepared using our recently developed Damp-Film method. The phase boundaries of liquid-ordered and liquid-disordered (Lo+Ld) coexisting region as well as the critical points were determined using video fluorescence microscopy. Within the phase coexisting regions, thermodynamic tie-lines were determined using a fluorescence assay. Our results show that adding 1 mol% of gramicidin produces significant and complex phase changes to the lipid bilayers: at some lipid compositions, gramicidin can induce lipid domains; at others, gramicidin completely abolish the phase separation; even if the phase separation is preserved, gramicidin significantly alters the lipid compositions of membrane domains and tie-lines. In the biological relevant critical region, these changes could be quite dramatic. We also measured gramicidin-A partition coefficients between coexisting Lo+Ld lipid phases. Away from the critical point, the coefficient is close to 2, indicating that gramicidin slightly prefers the disordered Ld lipid domains with smaller bilayer thickness. However, the partition coefficient continuously changes with lipid composition. Near the critical point, the partition coefficient approaches to the theoretical value of 1.

Actin Filaments Attachment to the Plasma Membrane Cause the Formation of Ordered Lipid Domains in Live Cells

The aim of this study was to investigate the relationship between ordered plasma membrane nanodomains and actin filaments using di-4-ANEPPDHQ and laurdan together with the reagents that affect actin filament dynamics in live Jurkat and primary T cells. The degree of lipid packing can be quantified using polarity sensitive membrane dyes such as laurdan and di-4-ANEPPDHQ. These two dyes display a red shift in their emission peaks for membranes in ld phase relative to lo phase. Laurdan is uncharged and can easily flip between two leaflets of the plasma membrane and we demonstrate that it reports equally on the two leaflets of the plasma membrane.

High‐Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells

Lipid analogues carrying three nitrilotriacetic acid (tris-NTA) head groups were developed for the selective targeting of His-tagged proteins into liquid ordered (lo ) or liquid disordered (ld ) lipid phases. Strong partitioning into the lo phase of His-tagged proteins bound to tris-NTA conjugated to saturated alkyl chains (tris-NTA DODA) was achieved, while tris-NTA conjugated to an unsaturated alkyl chain (tris-NTA SOA) predominantly resided in the ld phase. Interestingly, His-tag-mediated lipid crosslinking turned out to be required for efficient targeting into the lo phase by tris-NTA DODA. Robust partitioning into lo phases was confirmed by using viral lipid mixtures and giant plasma membrane vesicles. Moreover, efficient protein targeting into lo and ld domains within the plasma membrane of living cells was demonstrated by single-molecule tracking, thus establishing a highly generic approach for exploring lipid microdomains in situ.

High-Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells

Lipid analogues carrying three nitrilotriacetic acid (tris-NTA) head groups were developed for the selective targeting of His-tagged proteins into liquid ordered (lo) or liquid disordered (ld) lipid phases. Strong partitioning into the lo phase of His-tagged proteins bound to tris-NTA conjugated to saturated alkyl chains (tris-NTA DODA) was achieved, while tris-NTA conjugated to an unsaturated alkyl chain (tris-NTA SOA) predominantly resided in the ld phase. Interestingly, His-tag-mediated lipid crosslinking turned out to be required for efficient targeting into the lo phase by tris-NTA DODA. Robust partitioning into lo phases was confirmed by using viral lipid mixtures and giant plasma membrane vesicles. Moreover, efficient protein targeting into lo and ld domains within the plasma membrane of living cells was demonstrated by single-molecule tracking, thus establishing a highly generic approach for exploring lipid microdomains in situ.

Changes in Order Parameters Associated with Ceramide-Mediated Membrane Reorganization Measured Using pTIRFM

The enzymatic generation of ceramide has significant effects on the biophysical properties of lipid bilayers and can lead to the extensive reorganization of cell membranes. We have synthesized and characterized a headgroup-labeled fluorescent lipid probe (NBD-ceramide, NBD-Cer) and demonstrated that it can be used for polarized total internal reflection fluorescence microscopy experiments to probe changes in membrane order that result from ceramide incorporation. NBD-Cer measures significantly higher order parameters for the liquid-ordered (Lo) domains ([P2] = 0.40 ± 0.03) than for the liquid-disordered phase (Ld, fluid, [P2] = 0.22 ± 0.02) of phase-separated bilayers prepared from egg sphingomyelin, dioleolyphosphatidylcholine, and cholesterol mixtures. The probe also responds to changes in packing induced by the direct incorporation of ceramide or the variation in the ionic strength of the aqueous medium. Order parameter maps obtained after enzyme treatment of bilayers with coexisting Lo and Ld phases show two distinct types of behavior. In regions of high enzyme activity, the initial Lo/Ld domains are replaced by large, dark features that have high membrane order corroborating previous hypotheses that these are ceramide-enriched regions of the membrane. In areas of low enzyme activity, the size and shape of the Lo domains are conserved, but there is an increase in the order parameter for the initial Ld phase ([P2] = 0.30 ± 0.01). This is attributed to the incorporation of ceramide in the Lo domains with the concomitant expulsion of cholesterol into the surrounding fluid phase, increasing its order parameter.

Temperature dependence of diffusion in model and live cell membranes characterized by imaging fluorescence correlation spectroscopy

The organization of the plasma membrane is regulated by the dynamic equilibrium between the liquid ordered (Lo) and liquid disordered (Ld) phases. The abundance of the Lo phase is assumed to be a consequence of the interaction between cholesterol and the other lipids, which are otherwise in either the Ld or gel (So) phase. The characteristic lipid packing in these phases results in significant differences in their respective lateral dynamics. In this study, imaging total internal reflection fluorescence correlation spectroscopy (ITIR-FCS) is applied to monitor the diffusion within supported lipid bilayers (SLBs) as functions of temperature and composition. We show that the temperature dependence of membrane lateral diffusion, which is parameterized by the Arrhenius activation energy (EArr), can resolve the sub-resolution phase behavior of lipid mixtures. The FCS diffusion law, a novel membrane heterogeneity ruler implemented in ITIR-FCS, is applied to show that the domains in the So–Ld phase are static and large while they are small and dynamic in the Lo–Ld phase. Diffusion measurements and the subsequent FCS diffusion law analyses at different temperatures show that the modulation in membrane dynamics at high temperature (313K) is a cumulative effect of domain melting and rigidity relaxation. Finally, we extend these studies to the plasma membranes of commonly used neuroblastoma, HeLa and fibroblast cells. The temperature dependence of membrane dynamics for neuroblastoma cells is significantly different from that of HeLa or fibroblast cells as the different cell types exhibit a high level of compositional heterogeneity.

Antibody-Induced Acetylcholine Receptor Clusters Inhabit Liquid-Ordered and Liquid-Disordered Domains

The distribution of nicotinic acetylcholine receptor (AChR) clusters at the cell membrane was studied in CHO-K1/A5 cells using fluorescence microscopy. Di-4-ANEPPDHQ, a fluorescent probe that differentiates between liquid-ordered (Lo) and liquid-disordered (Ld) phases in model membranes, was used in combination with monoclonal anti-AChR antibody labeling of live cells, which induces AChR clustering. The so-called generalized polarization (GP) of di-4-ANEPPDHQ was measured in regions of the cell-surface membrane associated with or devoid of antibody-induced AChR clusters, respectively. AChR clusters were almost equally distributed between Lo and Ld domains, independently of receptor surface levels and agonist (carbamoylcholine and nicotine) or antagonist (α-bungarotoxin) binding. Cholesterol depletion diminished the cell membrane mean di-4-ANEPPDHQ GP and the number of AChR clusters associated with Ld membrane domains increased concomitantly. Depolymerization of the filamentous actin cytoskeleton by Latrunculin A had the opposite effect, with more AChR clusters associated with Lo domains. AChR internalized via small vesicles having lower GP and lower cholesterol content than the surface membrane. Upon cholesterol depletion, only 12% of the AChR-containing vesicles costained with the fluorescent cholesterol analog fPEG-cholesterol, i.e., AChR endocytosis was essentially dissociated from that of cholesterol. In conclusion, the distribution of AChR submicron-sized clusters at the cell membrane appears to be regulated by cholesterol content and cytoskeleton integrity.

Two-Dimensional Continuous Extraction in Multiphase Lipid Bilayers To Separate, Enrich, and Sort Membrane-Bound Species

A new method is presented to separate, enrich, and sort membrane-bound biomolecules based on their affinity for different coexisting lipid phases in a supported lipid bilayer using a two-dimensional, continuous extraction procedure. Analogous to classic liquid-liquid phase extraction, we created two distinct lipid phases in our planar membrane system: a liquid-ordered (l(o)) phase and a liquid-disordered (l(d)) phase arranged in parallel stripes inside a microfluidic device. Membrane-bound biomolecules in an adjacent supported lipid bilayer are convected in plane along the microfluidic channel and brought into contact with a different lipid phase using hydrodynamic force. A mixture of two lipid species, a glycolipid and a phospholipid, with known affinities for the two lipid phases employed here are used to demonstrate continuous extraction of the lipid-microdomain preferring glycolipid to the lo phase, while the phospholipid remains primarily in the ld phase. In this demonstration, we characterize the performance of this affinity-based separation device by building models to describe the velocity profile and transport in the two-phase coexistent membrane. We then characterize the impact of residence time on the extraction yield of each species. This new procedure sorts membrane species on the basis of chemical properties and affinities for specific lipid phases within a membrane environment near physiological conditions, critical for extending this method to the separation of lipid-linked proteins and transmembrane proteins while minimizing denaturation. This platform could facilitate the separation and identification of lipid membrane domain residents, or the characterization of changes in membrane affinity due to post-translational modifications or environmental conditions.

Effects of pyrenebutyrate on the translocation of arginine-rich cell-penetrating peptides through artificial membranes: Recruiting peptides to the membranes, dissipating liquid-ordered phases, and inducing curvature

Arginine-rich cell-penetrating peptides, including octaarginine (R8) and HIV-1 TAT peptides, have the ability to translocate through cell membranes and transport exogenous bioactive molecules into cells. Hydrophobic counteranions such as pyrenebutyrate (PyB) have been reported to markedly promote the membrane translocation of these peptides. In this study, using model membranes having liquid-ordered (Lo) and liquid-disordered (Ld) phases, we explored the effects of PyB on the promotion of R8 translocation. Confocal microscopic observations of giant unilamellar vesicles (GUVs) showed that PyB significantly accelerated the accumulation of R8 on membranes containing negatively charged lipids, leading to the internalization of R8 without collapse of the GUV structures. PyB displayed an alternative activity, increasing the fluidity of the negatively charged membranes, which diminished the distinct Lo/Ld phase separation on GUVs. This was supported by the decrease in fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH). Additionally, PyB induced membrane curvature, which has been suggested as a possible mechanism of membrane translocation for R8. Taken together, our results indicate that PyB may have multiple effects that promote R8 translocation through cell membranes.